directional cDNA cloning
Ron Goto x2832
rgoto at smtplink.coh.org
Thu Jan 4 18:09:06 EST 1996
In article <Pine.A32.3.91j.960104112441.120910A-100000 at homer15.u.washington.edu> Mayumi Yagi <myagi at u.washington.edu> writes:
>From: Mayumi Yagi <myagi at u.washington.edu>
>Subject: directional cDNA cloning
>Date: Thu, 4 Jan 1996 11:40:18 -0800
>Organization: University of Washington
>Message-ID: <Pine.A32.3.91j.960104112441.120910A-100000 at homer15.u.washington.edu>
>Content-Type: TEXT/PLAIN; charset=US-ASCII
>I'd like some advice on directional cDNA cloning. In the past I've
>successfully constructed nondirectional cDNA libraries, but for a variety
>of reasons we're considering doing a directional library in a homemade
>vector with EcoRI (5') and XhoI (3') sites. Stratagene sells a kit using
>precisely these enzymes, but before popping $600 I'd like to get some
>feedback both on the kit and the potential difficulties I might encounter
>in the protocol. Essentially, the protocol substitutes methyldC for dC in
>the cDNA synthesis reaction, the idea being to prevent cleavage by XhoI
>at any internal sites.
>I'd appreciate hearing from anyone who's used this kit, and any
>suggestions from others who have done directional cloning.
>Thanks in advance,
Yes, Mayumi I have recently used Stratagene cDNA kit to make my cDNA
libraries, IMHO it was very easy to use, direction were OK especially
since you have made cDNA library before (I made my first cDNA library
in 1989, also using stratagene kits) and my results were good (Ifound 9 full
length and 1- 3/4 length with gene 1 and 10 clones of similiar length for
gene 2 probably full length). Yes I said libraries, as you say $600, I made
4 different libraries (because screening takes time and kit are getting old)
The biggest thing about directional cloning is one; you can see with RE
same/similiar clones and two; if screening by antibody double you chances.
NO nothing with stratagene, just a customer. Also used their prepared vector.
Just what we have done, nothing special.
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